Effect of hibernation on sodium and chloride ion transport in isolated frog skin

The aim of the study was to evaluate the effect of hibernation on electrophysiological parameters of isolated frog skin under control incubation (Ringer solution) and after inhibition of Na+ and CI- transepithelial transport by application of amiloride and bumetanide. The transepithelial electrical potential difference (PD in mV) was measured before and after mechanical stimulation of isolated frog skin. The tissues were mounted in a modified Ussing chamber. The results revealed a reduced PD of frog skin during hibernation. In February, as compared with November, PD of frog skin incubated in Ringer solution decreased by about 50%. Hibernation also affected hyperpolarization (dPD) of frog skin after mechanical stimulation. In November and December, dPD was about 50% and 30% lower, respectively, compared with the subsequent two months of the experiment. The incubation of frog skin with amiloride, a sodium ion channel blocker, resulted in reduced values of all measured electrophysiological parameters irrespective of the phase of hibernation. After application of chloride ion transport inhibitor (bumetanide), the PD in November and December decreased compared with the control incubation by about 80% and 75%, while in January and February by about 40% and 25%, respectively. In January and February dPD increased by four times and three times as compared with November and December. Hibernation reduces net ion flow in isolated frog skin. During the initial period of hibernation the sensitivity of the skin to mechanical stimulation also decreases. Towards the end of hibernation, on the other hand, excitation of mechanosensitive ion channels takes place.     

Transepithelial transport of sodium and chloride ions in isolated skin of the frog,Rana esculenta L

Isolated frog skin, mounted in a Ussing apparatus, was investigated electrophysiologically. Application of amiloride, an inhibitor of sodium ion transport, and bumetanide, known to block the transport of chloride ions, revealed the effect of these ions on PD, both under control conditions and following mechanical stimulation. Under control conditions, mechanical stimulation of the skin caused hyperpolarization, i.e. a transient increase in the electrical potential difference. Preincubation in the presence of amiloride, or amiloride plus bumetanide, brought about both a decrease in electrical potential and an inhibition of the reaction upon stimulation. On the other hand, incubation with bumetanide resulted in a decrease in electrical potential, but did not affect the skin reaction after mechanical stimulation. The above results indicate that hyperpolarization of the frog skin following mechanical stimulation is caused by enhanced transepithelial transport of sodium ions which, in turn, is induced by stimulation of sensory receptors.     

Electrophysiological effect of ruthenium red and pH on the skin of the edible frog, Rana esculenta L

Amphibian skin is a sensitive interface between the organism and the environment. Metal ions from the external environment, some of them being trace elements, act on the amphibian skin. It had been shown that stimulation of tactile receptors affected Na+ transport in the frog skin and changed the potential difference, therefore the aim of this project was to study the effect of ruthenium complex, known as ruthenium red (RR), on the ion transport in this organ in vitro under control conditions, after mechanical stimulation and also in the presence of the Na+ transport inhibitor-amiloride. Three different concentrations of RR (0.12, 1.2, and 12.0 mM) in two different pH values (6.4 and 7.4) were studied in vitro in the Ussing apparatus. The measured electrophysiological parameters were the transepithelial electrical potential difference (PD) and the changes in PD after mechanical stimulation (dPD). The gentle mechanical stimulus was a jet of bath fluid from a peristaltic pump directed on the mucosal surface of isolated frog skin. After mechanical stimulation, transient hyperpolarization invariably occurred (dPD = 1.5 +/- 0.2 mV). In the presence of RR the hyperpolarization was smaller and this diminution was concentration dependent: 0.5 +/- 0.1 mV for 1.2 mM of RR and 0.1 +/- 0.1 mV for 1.2 mM of RR. At pH 6.4 the reactions of the skins on the mechanical and chemical stimuli were smaller, in the presence of amiloride disappearing completely, but after the washing away of amiloride from the experimental organ in pH 6.4 the action of RR was stimulatory. The natural defensive reactions of frog skin related to the ion transport and electrical potential difference are affected or disappear in the presence of ruthenium complex.     

The effect of serotonin on epinephrine modulation of transepithelial ion transport in isolated frog skin

The aim of this study was to examine the effect of serotonin and epinephrine on ion transport of isolated frog skin. The addition of serotonin after incubation in Ringer solution (RH), bumetanide (BUME), and after initial incubation in amiloride and subsequently in RH, reduced hyperpolarization and did not effect the mechanosensitivity of frog skin. Following incubation of the frog skin with amiloride (AMI), serotonin did not affect the value of hyperpolarization and increased mechanosensitivity. The addition of epinephrine (EPI) on frog skin incubated in RH and AMI did not affect hyperpolarization, but repeated application of this compound after serotonin increased hyperpolarization. After incubation with bumetanide, addition of EPI before and after application of serotonin did not affect the value of the examined parameters of the frog skin. Initial incubation with AMI and later in RH caused a drop in reaction to EPI and no effect on mechanosensitivity. Repeated addition of epinephrine in this group did not affect the reaction value, while it decreased the reaction value during mechanical stimulation. The experimental data presented in this study indicate that serotonin inhibits the sodium ion current. Epinephrine inhibits the chloride ion current, however, after the application of serotonin, EPI stimulates sodium ion transport.     

The effect of dimethyl sulphoxide on electron transport in chloroplasts

The effect of DMSO (dimethyl sulphoxide) on electron transport in chloroplast membranes has been studied. It has been found that concentrations of DMSO up to 20% (v/v) do not inhibit electron transport in freshly isolated chloroplasts, but that higher concentrations start to cause inhibition. However, in chloroplasts that have been aged for 8 to 24 hours by storage at 4 degrees C, the addition of DMSO at concentrations up to 20% causes stimulation of electron transport. Possible mechanisms for this effect are discussed.     

Effects of lysine-vasopressin (LVP) and 1-deamino-8-D-arginine-vasopressin (dDAVP) upon electrical potential, short-circuit current and transepithelial D.C. resistance of the frog skin

The synthetic analogue of vasopressin, 1-deamino-8-D-arginine-vasopressin (dDAVP), possesses a protracted antidiuretic activity while having practically no pressoric activity as compared to arginine-vasopressin (AVP) or lysine-vasopressin (LVP). The effects of LVP and dDAVP were studied on the frog skin (Rana temporaria) sodium transport as reflected by the short-circuit current (SCC) level, on an Ussing apparatus. The application two different equimolar doses of LVP or dDAVP (approx. 9.4 X 10(-8) mol X l-1 and 18.8 X 10(-8) mol X l-1 to the inner surface of the skin resulted in identical maximal increases of sodium transport. However, the maximum transport stimulation after the application of dDAVP was delayed by about 30 min as compared to the stimulation by LVP (P less than 0.01). In addition, a protracted recovery of SCC towards its original levels was observed in experiments with dDAVP application after the hormone removal (P less than 0.01). It is concluded that dDAVP stimulates Na+ transport through the frog skin despite its lacking pressoric activity. Thus, the natriferic activity of vasopressin is related to its antidiuretic rather than pressoric activity. Maximum increase in the sodium transport following dDAVP application was delayed and more protracted as compared to the effect of LVP.     

Oxygen consumption by frog skin and its isolated epithelial layers as a function of their sodium-transporting activity

The metabolic cost (in terms of oxygen consumption) of transcellular sodium transport was assessed on ventral frog skin and its isolated epithelial layers, by measuring the decrease in oxygen consumption by the tissue upon transient withdrawal of sodium from the outside solution. The same number of sodium ions was transported per molecule oxygen consumed by whole skin (17.4±2.3) and its isolated epithelium (17.3±2.4).The metabolic cost of sodium transport could not be estimated properly when this process was blocked by amiloride or ouabain, as these drugs were found to bring about an increase in oxygen consumption by the tissue when no sodium was available for transport.     

Oxygen consumption by frog skin and its isolated epithelial layers as a function of their sodium-transporting activity.

The metabolic cost (in terms of oxygen consumption) of transcellular sodium transport was assessed on ventral frog skin and its isolated epithelial layers, by measuring the decrease in oxygen consumption by the tissue upon transient withdrawal of sodium from the outside solution. The same number of sodium ions was transported per molecule oxygen consumed whole skin (17.4 +/- 2.3) and its isolated epithelium (17.3 +/- 2.4). The metabolic cost of sodium transport could not be estimated properly when this process was blocked by amiloride or ouabain, as these drugs were found to bring about an increase in oxygen consumpton by the tissue when no sodium was available for transport.     

Selective destruction of 5-hydroxytryptamine receptors by neuraminidase.

The responses of isolated frog skin to 5-hydroxytryptamine (increased active sodium transport and decreased passive chloride permeability) are diminished by incubation with the enzymes neuraminidase and N-acetylneuraminic acid aldolase but only in the absence of Ca2+ and presence of EDTA. The responses induced by oxytocin, adrenalin and aldosterone are unaffected by enzyme treatment.     

Alteration of membrane permeability by amphotericin B

Amphotericin B (AmB) increased unidirectional Na transport and net transcellular sodium movements across the skin of the frog, Rana pipiens, when added to the solution bathing the corium side, but not from the outer epidermal surface. The AmB response was prevented with pretreatment with amiloride, ouabain and mucosal sodium substitution. Alteration in pH markedly reduced the permeability changes induced by AmB. AmB did not interfere with the increase in sodium transport induced by antidiuretic hormone. The present study demonstrates that AmB interacts with the skin of the frog, Rana pipiens, from the corium side specifically increasing transepithelial sodium transport. The increase in transport apparently occurs through the existing sodium pathway.     

Selective destruction of 5-hydroxytryptamine receptors by neuraminidase

The responses of isolated frog skin to 5-hydroxytryptamine (increased active sodium transport and decreased passive chloride permeability) are diminished by incubation with the enzymes neuraminidase and N-acetylneuraminic acid aldolase but only in the absence of Ca²⁺ and presence of EDTA. The responses induced by oxytocin, adrenalin and aldosterone are unaffected by enzyme treatment.     

Effects of metabolic inhibitors and drugs on ion transport and oxygen consumption in isolated frog skin

Active ion (NaCl) transport across isolated frog skin is discussed in relation to sodium and potassium composition and to O(2) consumption of skin. A distinction is made between processes in skin related to "unidirectional active ion transport" and processes related to "maintenance electrolyte equilibrium;" i.e., ionic composition of skin. Several metabolic inhibitors were found that could be used in separating maintenance electrolyte equilibrium from unidirectional active ion transport. Fluoroacetate (up to 1 x 10(-2)M/liter) did not affect maintenance electrolyte equilibrium, but severely diminished the rate of active ion transport. This could also be accomplished with azide and diethyl malonate when 1 x 10(-3) molar concentrations were used. When applied in higher concentrations, these two inhibitors, and several others, diminished active ion transport, but this was associated with changes in maintenance electrolyte equilibrium (gain of Na(+) by and loss of K(+) from skin). Similar observations were made when skins were subjected to K(+)-deficient media. Mersalyl and theophylline, in low concentrations, stimulated active ion transport without leading to changes in maintenance electrolyte equilibrium. Inhibition of active ion transport was found accompanied by decrease, increase, and unaltered over-all O(2) consumption, depending on the kind of chemical agent used. A provisional scheme of the mechanism of unidirectional active ion transport is proposed. It is conceived as a process of metabolically supported ion exchange adsorption, involving a carrier, forming complexes with K(+) and Na(+), a trigger, K(+) ions, and two spatially separated metabolic pathways.     

The methionine sulphoxide reductase activity of the yeast dimethyl sulphoxide reductase system

Abstract Glycyl- l -methionine sulphoxide and N-acetyl- l -methionine sulphoxide were less effective inhibitors of the dimethyl sulphoxide (DMSO) reductase activity of Saccharomyces cerevisiae NCYC240 than was l -methionine (±)-sulphoxide. Methionine sulphoxide reductase and DMSO reductase activities from crude extracts co-purified over five purification steps and the two activities eluted from columns in exactly the same fractions. Both activities were sensitive to the same inhibitors. It was concluded that: (1) the DMSO reductase activity of S. cerevisiae is a property of a methionine sulphoxide reductase different from that of Black et al. [J. Biol. Chem. 235 (1960) 2910–2916], and (2) free methionine sulphoxide rather than peptidyl methionine sulphoxide is probably the enzyme's true substrate.     

Dimethyl sulphoxide reduction by micro-organisms.

Dimethyl sulphoxide (DMSO) was reduced to dimethyl sulphide by a wide variety of micro-organism, including prokaryotes and eukaryotes, aerobes and anaerobes. Dimethyl sulphone was not reduced by any of the organisms tested. Cell-free extracts of Escherichia coli reduced DMSO using reduced pyridine nucleotides as electron donors. Activity was greater in anaerobically grown cells than in those grown aerobically. Two other sulphoxides, methionine sulphoxide and tetramethylene sulphoxide, substantially inhibited DMSO reduction by extracts. Mutants of E. coli, which were unable to reduce biotin sulphoxide to biotin, were tested for their ability to reduce DMSO in whole cells and extracts. These mutants were in four different gene loci, bisA to bisD. DMSO reductase activity of the mutants was generally less than that of the wild-type strain, and activity depended upon the gene locus involved, the growth medium and the growth conditions. Only the bisA mutant had very low activity under all conditions. All of the bis mutants were able to grow using methionine sulphoxide as a sulphur source, indicating that biotin sulphoxide and methionine sulphoxide are reduced by different enzyme systems. DMSO may be reduced by both of these enzyme systems.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) was found not to inhibit active sodium transport across the isolated frog skin when added in 10^–3 m concentration to the basal-lateral surface. The same Cd⁺⁺ concentration similarly had no effect on Na⁺ transport across the isolated epithelial cell layer from the frog skin, although this dose of Cd⁺⁺ did inhibit Na⁺ transport across the frog urinary bladder and large intestine. When 10^–3 m Cd⁺⁺ was added to the apical surface of the isolated frog skin or to the isolated epithelial cells from the frog skin, sodium transport was reversibly stimulated, in contrast to the irreversible inhibition noted above. If equimolar cysteine was added with Cd⁺⁺ to the apical surface of the skin, however, active Na⁺ transport was irreversibly inhibited. In conjunction with the inhibition produced by equimolar Cd⁺⁺ and cysteine, isotopic Cd⁺⁺ permeation into the tissue was three times higher when added with cysteine than in the absence of cysteine. Thus, the effects of Cd⁺⁺ on epithelial Na⁺ transport is variable according to the epithelium studied and the presence of potential carrier molecules.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Opposite actions of different doses of arginine-vasotocin and 1-deamino-arginine-vasotocin on sodium ion transport in skin of the frog Rana temporaria

Active transport of sodium ions across the isolated abdominal skin of the frog Rana temporaria after application of arginine-vasotocin (AVT) and 1-deamino-arginine-vasotocin (1dAVT) was studied by measurement of the short-circuit current (SCC). The maximal increase in the SCC values (26 and 19 microA/cm2) was observed after addition of 10 nM AVT or 100 nM 1dAVT, respectively, to the frog skin basal surface. An increase of concentration of AVT to 100 nM and of IdAVT to 1 microM terminated the sodium transport in the frog skin. A preliminary addition of an antagonist of arginine-vasopressin V1a-receptors to the Ringer's solution at the frog skin basal surface led to a rise in the SCC values in response to administration of ineffective doses of AVT or 1dAVT. V2-receptor antagonists did not affect the frog skin reaction to administration of these doses of AVT or IdAVT.     

An enzymatic ion exchange model for active sodium transport

An enzymatic ion exchange model for active sodium transport is described. Kinetic equations relating net flux to time, and to concentration difference across the actively transporting membrane are derived. The second of these equations is tested, using the isolated frog skin in the "short-circuit" apparatus of Ussing. Reasonable linearity, as predicted by this equation, is observed. The passive permeability coefficient for Na⁺, is calculated as 5.3 x 10^-4 ± 5.3 x 10^-4 cm./hr. If cholinesterase is assumed to be the enzyme responsible for transport, the activity required to account for the observations reported here is 17.7 x 10^-4 mmoles/cm.²/hr.     

SOME MORPHOLOGICAL ASPECTS OF ACTIVE SODIUM TRANSPORT* : The Epithelium of the Frog Skin

A method was experimentally tested which allows simultaneous morphological and bioelectrical studies of a tissue that performs active sodium transport, i.e., the isolated, surviving frog skin. In a four cell lucite chamber with four separate electric potential and current circuits, skin specimens for morphological observation (light and electron microscopy) were fixed in situ in well-defined functional states. The rate of active sodium transport through the epithelium of Rana temporaria skin was modified by changing the strength of the electric current passed through the specimens. A marked, reversible swelling of the outermost layer of the stratum granulosum was observed during short circuiting of the skin compared to the homogeneous appearance of the epithelium under open circuit conditions. Doubling the ingoing current led to an additional small increase of the swelling or the appearance of islets of cell necrosis in the same layer. There were signs of a slight shrinkage of the underlying cell layers. The observations are discussed in the light of previous bioelectrical and morphological observations.